title: Screening and characterization of a cellulase gene from the gut microflora of abalone using metagenomic library.
abstract: A metagenomic fosmid library was constructed using genomic DNA isolated from abalone intestine. Screening of a library of 3,840 clones revealed a 36 kb insert of a cellulase positive clone (pAMHElO). A shotgun clone library was constructed using the positive clone (pAMHElO) and further screening of 3,840 shotgun clones with an approximately 5 kb insert size using a Congo red overlay revealed only one cellulase positive clone (pAMHL9). The pAMHL9 consisted of a 5,293-bp DNA sequence and three open reading frames (ORFs). Among the three ORFs, cellulase activity was only shown in the recombinant protein (CelAMll) coded by ORF3, which showed 100% identity with outer membrane protein A from Vibrio alginolyticus 12G01, but no significant sequence homology to known cellulases. The expressed protein (CelAMll) has a molecular weight of approximately 37 kDa and the highest CMC hydrolysis activity was observed at pH 7.0 and 37°C. The carboxymethyl cellulase activity was determined by zymogram active staining and different degraded product profiles for CelAMll were obtained when cellotetraose and cellopentaose were used as the substrates, while no substrate hydrolysis was observed on oligosaccharides such as cellobiose and cellotriose.
authors.affiliation: Department of Food Science and Technology and Functional Food Research Center, Chonnam National University, Gwangju, 500-757, Republic of Korea.
authors.forename: Duwoon
authors.initials: D
authors.lastname: Kim
authors.forename: Se-Na
authors.initials: SN
authors.lastname: Kim
authors.forename: Keun Sik
authors.initials: KS
authors.lastname: Baik
authors.forename: Seong Chan
authors.initials: SC
authors.lastname: Park
authors.forename: Chae Hong
authors.initials: CH
authors.lastname: Lim
authors.forename: Jong-Oh
authors.initials: JO
authors.lastname: Kim
authors.forename: Tai-Sun
authors.initials: TS
authors.lastname: Shin
authors.forename: Myung-Joo
authors.initials: MJ
authors.lastname: Oh
authors.forename: Chi Nam
authors.initials: CN
authors.lastname: Seong
mesh_terms: D000818:Animals; D002266:Carboxymethylcellulose Sodium / Q000378:metabolism; D002480:Cellulase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002482:Cellulose / Q000031:analogs & derivatives / Q000378:metabolism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D041981:Gastrointestinal Tract / Q000382:microbiology*; D049851:Gastropoda / Q000382:microbiology*; D015723:Gene Library; D006863:Hydrogen-Ion Concentration; D054892:Metagenome; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009844:Oligosaccharides / Q000378:metabolism; D016366:Open Reading Frames; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature; D013780:Tetroses / Q000378:metabolism; D044149:Vibrio alginolyticus / Q000235:genetics
chemical_list: D009844:Oligosaccharides; D013780:Tetroses; C019193:maltopentaose; C048468:cellotetraose; D002482:Cellulose; D002480:Cellulase; D002266:Carboxymethylcellulose Sodium